Alternative rownstream roles for Ste2p and an α-arrestin in sacccharomyces cerevisiae mating

Ste2p and Ste3p are well-characterized yeast pheromone G-protein Coupled Receptors (GPCR) those are involved in the signaling of mating responses that lead to cell fusion. Their signaling–associated interactions with G-protein/MAPK signal transduction machinery are well established, homologous to those in mammalian systems, and serve as a simplified model system in GPCR research. While the arrestin- mediated biased signaling mechanism of mammalian GPCR has not been discovered for the pheromone receptors, a recent demonstration of α-arrestins being involved in the internalization of the pheromone GPCR, Ste2p was reported. The present study was designed to reevaluate and extend the alternate functionality for pheromone receptors and to determine the role of yeast arrestins in the yeast mating. Specific residues in the TM6 of Ste2p exhibiting strong mating and constitutive MAPK signaling were combined and investigated in terms of their effect on MAPK signal transduction leading to cell cycle arrest as well as their impact on downstream mating projection formation and zygote formation events. Our findings indicate that Ste2p possess as specific residues that govern its relative bias for mediating MAPK signaling or mating events. Relative dose response experiments accounting for systemic and observation bias for these mutations yielded evidence of mutational-derived functional biases for Ste2p and further validated the alternate pheromone dependent functionalities for Ste2p. Further, arrestin knockout and knock-in studies showed that Art1 (Ldb19) is selectively involved in the regulation of zygote formation but not MAPK signal transduction following the binding of ligand to Ste2p receptors. In addition, ligand stimulated selective localization of Art1 (Ldb19) to the mating projection, implicating it in the regulation of downstream mating functionalities. Overall, while leaving the full mechanism of alternate/biased Ste2p signaling to be elucidated, these results highlight the possibility of continued relevance of the yeast pheromone-mating pathway as a simplified model for GPCR research in the context of arrestin-mediated biased GPCR signaling